Changes of the smooth endoplasmic reticulum induced by rifampicin in human and guinea-pig hepatocytes

Rifampicin induces a proliferation of the smooth endoplasmic reticulum in guinea-pig and human hepatocytes. This may support the hypothesis of enhancement of drug-metabolizing enzymes induced by the drug. However, the pattern of proliferation is not similar in man and in guinea-pig hepatocytes. Some caution is needed in the study of enzyme induction in man and in extrapolations from animal to human data.     

Immunolocalization of a novel cholesteryl ester hydrolase in the endoplasmic reticulum of murine and human hepatocytes

We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissue-restricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism.     

Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts

Tocopherols are nonpolar compounds synthesized and localized in plastids but whose genetic elimination specifically impacts fatty acid desaturation in the endoplasmic reticulum (ER), suggesting a direct interaction with ER-resident enzymes. To functionally probe for such interactions, we developed transorganellar complementation, where mutated pathway activities in one organelle are experimentally tested for substrate accessibility and complementation by active enzymes retargeted to a companion organelle. Mutations disrupting three plastid-resident activities in tocopherol and carotenoid synthesis were complemented from the ER in this fashion, demonstrating transorganellar access to at least seven nonpolar, plastid envelope-localized substrates from the lumen of the ER, likely through plastid:ER membrane interaction domains. The ability of enzymes in either organelle to access shared, nonpolar plastid metabolite pools redefines our understanding of the biochemical continuity of the ER and chloroplast with profound implications for the integration and regulation of organelle-spanning pathways that synthesize nonpolar metabolites in plants.     

Iron and uroporphyrin in hepatocytes of inbred mice in experimental porphyria: a biochemical and morphological study

Hexachlorobenzene-induced porphyria is iron dependent and characterized by the decreased activity of uroporphyrinogen decarboxylase and the accumulation of porphyrins in the liver. To examine the relationship between iron and porphyrins in liver tissue, we performed a biochemical and morphological (histological, ultrastructural and morphometrical) study in the livers of C57BL/10 mice. Mice were treated with hexachlorobenzene, iron dextran or the combination of hexachlorobenzene and iron dextran. An accumulation of porphyrins and an increased total iron content were found not only in the livers of mice treated with hexachlorobenzene and iron dextran but also in mice treated with iron dextran alone. In contrast, the amount of porphyrins was only slightly increased in the livers of mice treated with hexachlorobenzene alone. Needle-like structures, representing uroporphyrin crystals, were observed, histologically and ultrastructurally, in hepatocytes of mice treated with hexachlorobenzene and iron dextran and with iron dextran alone. Uroporphyrin crystals and ferritin iron were found in the same hepatocyte. A single uroporphyrin crystal, surrounded by ferritin iron, was observed in a hepatocyte of a mouse treated with hexachlorobenzene alone. Both in the livers of mice treated with hexachlorobenzene and iron dextran and in the livers of mice treated with iron dextran alone, morphometrical analysis showed that an increased area fraction of uroporphyrin crystals was associated with an increased area fraction of ferritin iron in hepatocytes. Conclusions: In C57BL/10 mice, experimental porphyria can be induced by iron overload alone; uroporphyrin crystals and ferritin iron are located in the same hepatocyte; and the morphological co-occurrence of uroporphyrin crystals and ferritin iron in hepatocytes suggests a role for iron (as ferritin) in the pathogenesis of porphyria.     

内质网应激与肝损伤研究进展

肝脏疾病组织学损伤,在细胞水平上可分为坏死和凋亡.近年来,大量研究表明,应激可能是肝损伤发生过程中的重要环节,如内质网应激、线粒体应激和氧化应激等,其中内质网应激是医学研究的一个新热点.本文将以内质网应激为切入点,探讨内质网应激的作用功能,内质网应激对细胞凋亡的影响,以及内质网应激与各种肝损伤之间的关系并对之进行综述.     

Correlation between biochemical and morphological repair in rabbit lungs after elastase injury

The structural and elastic properties of the lung depend primarily on the concentration in connective tissue of proteins, collagen and elastin. The relationships between biochemical and morphological aspects of pulmonary damage were studied in 50 male New Zealand rabbit’s, randomly divided into two groups of 25 animals, instilled with 245 IU of pig pancreatic elastase solution or physiological saline. Animals were killed at 8, 15, 30, 60 and 90 days after treatment. The changes in pulmonary morphology and in collagen and elastin content have been studied chemically and by light microscopy. The left lungs were used for biochemical determinations of insoluble elastin and total, insoluble and salt-extractable collagens. Morphometric measurements (mean linear intercepts or Lm) and histologic evaluations were performed on right lungs. Three months after instillation, the elastase treated lungs showed a minimal degree of alveolar wall rupture, and no significant difference in Lm from the control group. Quantitative restoration of normal biochemical parameters preceded connective tissue repair. Statistical evaluations showed significative correlations among Lm, total collagen (p < 0.01) and insoluble elastin/dry weight (p < 0.01). No significant differences between Lm and insoluble elastin, salt-extractable collagen or total collagen/dry weight were found.     

A correlative study between glucocorticoid receptor levels in human mononuclear leukocytes and biochemical data in Cushing's disease

We determined glucocorticoid receptors in human mononuclear leukocytes in 9 patients with Cushing's disease, in order to correlate them with laboratory data. Receptors were measured by a whole-cell assay method, after incubation with [3H]-dexamethasone in the presence or absence of excess unlabelled hormone. In Cushing's disease, there were 4425 +/- 364 sites/cell (N = 9), similar to in the controls: 4473 +/- 476 (N = 10); average Kd was 2.42 +/- 0.52 nmol/l (N = 3) similar to in the controls: 2.0 +/- 0.20 nmol/l (N = 3). In Cushing's patients we found significant negative correlations between basal glucocorticoid receptors and: 1) morning blood cortisol (r = -0.67, P less than 0.05), and 2) 17-ketogenic steroids after 2 mg of dexamethasone (r = -0.85, P less than 0.01). No correlations were observed with afternoon blood cortisol, free urinary cortisol, basal and post-8-mg dexamethasone 17-ketogenic steroids, TRH-TSH area, urinary calcium, plasma glucose, or systolic blood pressure. Conclusions: In Cushing's disease, a subtle receptor down-regulation may exist, as suggested by the inverse relationship between glucocorticoid receptors and morning blood cortisol. Secondly, the relationship between basal receptors and 17-ketogenic steroids after 2 mg of dexamethasone suggests that glucocorticoid receptors in human mononuclear leukocytes could reflect the sensitivity of the nervous system-pituitary-adrenal axis to dexamethasone inhibition.     

The endoplasmic reticulum-sarcoplasmic reticulum connection: distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers.

The skeletal muscle sarcoplasmic reticulum (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BiP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fractions revealed the presence of BiP in fast- and slow-twitch muscles of the rabbit as well as in rat and chicken muscles. Analyses of purified SR subfractions, together with cryosection immunofluorescence and immunogold labeling, revealed BiP evenly distributed within the longitudinal SR and the terminal cisternae. Within the terminal cisternae BiP appeared not to be mixed with calsequestrin but to be distributed around the aggregates of the latter Ca2+ binding protein. Of the various membrane markers only calnexin (91 kDa) was found to be distributed within both SR subfractions, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a specialized ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The differential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system.     

肝癌细胞蛋白质组在DTT诱导内质网应激条件下的表达

目的:研究内质网应激条件下肝癌细胞SMMC-7721蛋白质组的表达,为人类肝细胞癌的诊治提供新的靶点.方法:培养肝癌细胞株SMMC-7721,随机分为两组,即实验组和对照组.实验组加入二硫苏糖醇(DTT,2.5 mmol/L),对照组加入等量的培养基,裂解细胞,提取全细胞蛋白.双向电泳(2-DE)分离,Image Ma...     

Bromocriptine treatment reduces the cell size in human macroprolactinomas: a morphometric study

A morphological study was carried out on eight macroprolactinomas surgically removed from untreated patients and on nine macroprolactinomas removed from patients treated with bromocriptine (Brc; seven from patients treated for 6 weeks and two from patients treated for 1 yr). All treated patients had both serum PRL levels and tumor size (by computed tomographic scan) reduced. The study was carried out using light, immunofluorescence, and electron microscopic techniques and electron microscopic morphometry. By immunofluorescence microscopy, all tumors consisted of cells positive for PRL. In some treated tumors, the fluorescence was more marked than in untreated tumors. By light and electron microscopy and by morphometry, significant reductions in cell size were observed in the adenomas from Brc-treated patients. Both the cytoplasm and the nucleus shrank, but the reduction of the cytoplasm was much greater than that of the nucleus. The shrinkage of the cytoplasm was a consequence of marked involution of the rough endoplasmic reticulum and the Golgi complex in Brc-treated tumors. This effect may be related to inhibition of PRL synthesis by the drug. In some tumors from treated patients, the number of secretory granules was increased. No differences were observed between tumors from patients treated for 6 weeks and those from patients treated for 1 yr. The observed reduction in cell size by Brc could explain, at least in part, the well known size reduction of PRL-secreting adenomas in patients treated with the drug.     

Quantitative analysis of smooth endoplasmic reticulum proliferation in hepatocytes of early postnatal and adult mice treated with phenobarbital

The smooth endoplasmic reticulum in hepatocytes of early postnatal and adult ddY male mice injected with phenobarbital was analyzed stereologically. The animals received daily intraperitoneal injections of phenobarbital and were killed about 24 h after the last injection. In early postnatal animals (injected with 35 mg/kg of the drug at days 1 and 2 after birth, or injected with 35 mg/kg at day 2 and with 50 mg/kg at days 3 and 4 after birth), the smooth endoplasmic reticulum proliferated both in perivenular and periportal hepatocytes; there was no significant difference in amount of smooth endoplasmic reticulum between the cells of the two zones. In adult animals, however, proliferation occurred only in perivenular cells after the administration of 35 mg/kg of phenobarbital for 2 days, and predominantly in perivenular cells even after the administration of 150 mg/kg of the drug for 7 days. These results suggest that the conspicuous proliferation of the smooth endoplasmic reticulum in perivenular hepatocytes in response to phenobarbital administration, which is characteristic of adult liver, does not occur in early postnatal mice but becomes manifest during postnatal development.     

间歇低氧与内质网应激关系的研究进展

内质网是蛋白质折叠和转运的重要场所,机体受到多种刺激可引起细胞内一系列反应,称为内质网应激(ERS)。OSAHS特征性的间歇低氧可引起 ERS,而 ERS在 OSAHS造成多系统损害中发挥的作用受到越来越多的关注,本文就间歇低氧与 ERS相互关系作一综述。     

内质网应激与酒精性心肌病的关系研究进展

近年来,随着酒精性饮品的摄入增加,酒精性心肌病(ACM)的发病率和死亡率也增加。研究发现ACM发病涉及多层次和水平的调控。而且,细胞信号调控的关键场所内质网应激(ERS)在ACM中的作用受到重视。因此,本文对ACM的概述、发病机制以及ERS在酒精所致心肌损伤中的作用进行简要综述。     

Age-associated changes in rat hepatocytes: An early decline in synthesis of a specific protein by endoplasmic reticulum

Isolated hepatocytes from rats of disparate age were compared for their capacity to incorporate radioisotopic leucine into proteins of subcellular fractions. Determinations of isotope ratios of component microsomal proteins derived from such experiments reveal an age-associated decline in incorporation of leucine into a specific protein of the endoplasmic reticulum. The onset of this decline is detectable at as early as 6 months of age and the depression of synthesis is complete in animals 20 months of age or older.     

Endogenous peroxidase in the nuclear envelope and endoplasmic reticulum of human monocytes in vitro: association with arachidonic acid metabolism

The development of peroxidase (PO) reaction in the nuclear envelope (NE) and endoplasmic reticulum (ER) of monocytes differentiating in vitro and its relationship with arachidonic acid metabolism were studied. The PO, as visualized by the diaminobenzidine (DAB) technique, appeared in the NE and ER of the majority of monocytes within 24 hours of culture, with a substantial decrease thereafter. The influence of three major groups of agents--inhibitors of PO, of prostanoids, and of protein biosynthesis--upon the development of the PO reaction was examined. When aminotriazole, a PO inhibitor, was added to the culture medium, the appearance of PO was suppressed in the monocytes. The cyclooxygenase blocker, indomethacin, however, did not influence the development of PO. Also the blockers of protein synthesis, puromycin, cycloheximide, and actinomycin D, did not affect the appearance of PO. The prostanoids released from the monocytes, ie, prostaglandin E and thromboxane B2, were determined by radioimmunoassay and showed a time sequence of secretion that corresponded to the appearance of PO in the cells: a marked increase within the first 24 hours with a substantial decrease thereafter. The presence of the PO inhibitors aminotriazole and sodium azide in the culture medium produced a suppression of prostanoid release from the monocytes comparable with that of indomethacin. The data suggest that the PO in the NE and ER of differentiating monocytes in vitro (1) is associated with arachidonic acid metabolism, and (2) is not formed by de novo protein synthesis but rather by an activation process.